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Breakthrough Articles

'Breakthrough Articles’ present high-impact studies answering long-standing questions in the field of nucleic acids research and/or opening up new areas and mechanistic hypotheses for investigation. These articles are chosen by the Editors on the recommendation of Editorial Board Members and Referees. Articles are accompanied by a brief synopsis explaining the findings of the paper and where they fit in the broader context of nucleic acids research. Breakthrough Articles replace the older 'Featured Articles’, which have been discontinued. They represent the very best papers published at NAR.

Feature Articles

A dimeric state for PRC2
Davidovich C, Goodrich KJ, Gooding AR, Cech TR.
Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Here we provide the first direct evidence, using multiple independent approaches, that the four-subunit and five-subunit PRC2 complexes are dimers, including eight and ten protein subunits, respectively. We are further showing that each PRC2 dimer binds a single RNA, but a single RNA can bind multiple PRC2 dimers. Our findings lead to a new molecular mechanism that can explain how a single RNA transcript can nucleate repression by binding multiple PRC2 complexes, through PRC2 dimerization and cooperative RNA binding. Free Full Text

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice
Prakash TP, Graham MJ, Yu J, Carty R, Low A, Chappell A, Schmidt K, Zhao C, Aghajan M, Murray HF, Riney S, Booten SL, Murray SF, Gaus H, Crosby J, Lima WF, Guo S, Monia BP, Swayze EE, Seth PP.
Inefficient delivery of antisense oligonucleotides to target tissues can limit the efficacy of nucleic acid drugs. The authors demonstrate that a GalNac modification can enhance the potency and duration of action of two different antisense oligonucleotides targeting important biomedical targets human apolipoprotein C-III and transthyretin. The relatively straightforward chemical modification revealed in this paper improves potency by approximately 10-fold. By reducing the amount of drug needed cost will be lowered and the potential for side effects probably reduced. While the GalNac conjugates will only improve potency in the liver, one can imagine using similar modification strategies to assist uptake by other organs. The liver is already a focus for antisense therapy, so there is likely to be a significant impact on clinical development in the near term. Free Full Text

PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1
Langelier MF, Riccio AA, Pascal JM.
This paper describes details of a mechanism by which two DNA damage-dependent enzymes (termed 'Poly-ADP-ribose polymerases' or 'PARPs') respond to DNA damage, through specific activation in response to generation of free 5' phosphate groups at the sites of DNA strand breaks. The study demonstrated that two PARP family members (PARP-2 and PARP-3) may exploit these interactions to act at late stages of the repair process for such strand breaks, perhaps by facilitating subsequent ligation of the cleaved DNA ends. These results provide new insight into mechanisms by which the fidelity of a genome (which is under constant assault by a wide variety of internal and external DNA damaging agents in the cell) is maintained through the action of protective repair molecules. Free Full Text

Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters
Loughery J, Cox M, Smith LM, Meek DW.
This study focuses on a key aspect of the mechanism by which the p53 tumour suppressor protein, which is crucial in preventing cancer cell survival, is activated upon receiving an appropriate stimulus. The authors show that a specific reversible chemical modification of p53 (phosphorylation) previously thought to have a limited involvement, is essential for p53 function and is required for the ability of p53 to activate the cellular machinery that leads to the manufacture of the cellular components needed to combat cancer-associated changes in the cell. This information extends our knowledge of the basic principles by which p53 functions and is controlled. Free Full Text

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment
Taniguchi I, Mabuchi N, Ohno M.
These studies address a long standing question about the export of HIV-1 RNAs, establish a previously unrecognized function of the viral Rev protein and in so doing suggest potential new targets for anti-retroviral drugs. Free Full Text

Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification
Neumann P, Lakomek K, Naumann PT, Erwin WM, Lauhon CT, Ficner R.
More than 100 chemically modified nucleosides have been identified in tRNAs. One of them, 4-thiouridine, is found at position 8 of all tRNAs in bacteria and archaea. It stabilizes the tRNA fold and, in addition, serves as UV light sensor inducing a cellular response to UV radiation. The structural basis for the exclusive C4-thiolation of U8 has been elucidated by the crystal structure analysis of a 4-thiouridine synthetase - RNA complex. The structure revealed that the enzyme employs a molecular ruler measuring the distance from the 3’-CCA end, which is common to all tRNAs, to the site of modification. Free Full Text

Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation
Aik W, Scotti JS, Choi H, Gong L, Demetriades M, Schofield CJ, McDonough MA.
It is now widely recognised that nucleic acids can be marked with chemical groups, or ‘tagged’, in the form of methylation. Like an accented letter of the alphabet (e.g. á), these marks change the way the genetic code is ‘read’, resulting in a change in gene expression. Enzymes that tag nucleic acids with methyl-mark 'accents' are called methyltransferases and can be thought of as ‘writers’. Enzymes that remove methyl-mark accents are called demethylases or ‘erasers’. This dynamic nucleic acid modification process provides for a complex regulatory mechanism of gene expression known as epigenetics. ALKBH5 is one of two enzymes known to ‘erase’ N6-methyladenine (m6A) on RNA. Recently discovered cellular and physiological roles (e.g. obesity, fertility and circadian rhythm) for RNA m6A have been exciting researchers in all aspects of biology. The crystal structure presented here reveals the mechanisms of nucleic acid recognition and demethylation of ALKBH5, laying a foundation for further mutational and inhibition studies. Free Full Text

Three-tiered role of the pioneer factor GATA2 in promoting androgen-dependent gene expression in prostate cancer
Wu D, Sunkel B, Chen Z, Liu X, Ye Z, Li Q, Grenade C, Ke J, Zhang C, Chen H, Nephew KP, Huang TH, Liu Z, Jin VX, Wang Q.
Though the importance of GATA family pioneer factors in facilitating ligand-inducible nuclear receptor activity is well appreciated, the mechanism of their action is not fully understood. We show in prostate cancer cells, in which disease-related gene expression is stimulated by male hormones acting through the androgen receptor (AR), that GATA2 takes a three-pronged approach in advance of hormone treatment to prime for AR-driven gene expression. Our findings establish a hormone-independent role for GATA2 in determining chromatin accessibility of gene regulatory elements and as a component of transcription-activating chromatin loops within AR target gene loci. Free Full Text

A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis
Pimentel H, Parra M, Gee S, Ghanem D, An X, Li J, Mohandas N, Pachter L, Conboy JG.
By comparing alternative pre-mRNA splicing in five morphologically distinct populations of successively more differentiated erythroblasts, the authors characterized a robust and dynamic erythroid alternative pre-mRNA splicing program. Erythroblast alternative splicing is particularly enriched in genes controlling cell cycle, organelle organization, chromatin function, and RNA processing. Differentiation stage-specific changes in splicing are executed in concert with, and may help drive, major cellular remodeling in the last stages before enucleation. A subset of splicing switches introduce premature translation termination codons, indicating that alternative splicing-coupled nonsense-mediated-decay (AS-NMD) contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. Free Full Text

The Vertebrate Genome Annotation browser 10 years on
Harrow JL, Steward CA, Frankish A, Gilbert JG, Gonzalez JM, Loveland JE, Mudge J, Sheppard D, Thomas M, Trevanion S, Wilming LG.
The Vertebrate Genome Annotation (VEGA) browser is a database for viewing manually curated genes on high quality genome sequence. It has recently been updated with an improved user interface to make navigating the different datasets easier. We have increased the number of annotated genomes to include human, mouse, zebrafish, rat and pig whole genomes, plus small regions of medical interest from organisms as diverse as gorilla, wallaby and Tasmanian devil. Free Full Text

TFBSshape: a motif database for DNA shape features of transcription factor binding sites
Yang L, Zhou T, Dror I, Mathelier A, Wasserman WW, Gordân R, Rohs R.
Our current understanding of DNA binding specificities of transcription factors (TFs) mainly results from nucleotide sequence analysis. However, proteins recognize DNA as a three-dimensional object. Thus, an improved comprehension of DNA binding specificity must be based on DNA structure. The TFBSshape database augments nucleotide sequence motifs with heat maps and quantitative predictions of DNA shape features for 739 TF datasets from 23 different species. Sequence information analyzed by TFBSshape is collected from the motif databases JASPAR and UniPROBE. The structural characterization of transcription factor binding sites (TFBSs) can aid users in gaining further insights into the mechanisms underlying TF-DNA recognition. Free Full Text

Cycling of the E. coli lagging strand polymerase is triggered exclusively by the availability of a new primer at the replication fork
Yuan Q, McHenry CS.
Two models have been proposed for triggering release of the lagging strand polymerase at the E. coli replication fork, enabling cycling to the primer for the next Okazaki fragment—either collision with the 5’-end of the preceding fragment (collision model), or synthesis of a new primer (signaling model). Specific perturbation of lagging strand elongation on minicircles confirmed the signaling model and ruled out the collision model. It was demonstrated that the presence of a primer, not primase per se, provides the cycling signal and that the rate of lagging strand elongation is faster than the leading strand. Free Full Text

The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification
Dreveny I, Deeves SE, Fulton J, Yue B, Messmer M, Bhattacharya A, Collins HM, Heery DM.
New crystal structures reveal for the first time that the Double PHD Finger domain of the chromatin regulator MOZ/MYST3 can induce an alpha helical fold of the N-terminal tail of Histone H3. This enables MOZ to sample for the presence of existing histone modifications, and promotes acetylation of H3K14 by the MYST domain. A conserved double glycine hinge motif in the H3 tail is essential to allow the acetylated H3K14 residue to dock with the DPF domain. The study provides important new insights into how a chromatin regulator manipulates and modifies histone tail structure to dictate its own residency on chromatin. Free Full Text

Co-regulated gene expression by estrogen receptor-α and liver receptor homolog-1 is a feature of the estrogen response in breast cancer cells
Lai CF, Flach KD, Alexi X, Fox SP, Ottaviani S, Thiruchelvam PT, Kyle FJ, Thomas RS, Launchbury R, Hua H, Callaghan HB, Carroll JS, Charles Coombes R, Zwart W, Buluwela L, Ali S.
Most breast cancers are estrogen responsive and require the Estrogen receptor-α (ERα) transcription factor. By mapping DNA binding in the genome of breast cancer cells, we show that a distantly related transcription factor, LRH-1, co-regulates target genes with ERα, through the use of overlapping DNA binding motifs. Remarkably, LRH-1 promotes changes in chromatin to facilitate ERα recruitment, thereby leading to a co-operative regulation of gene expression. These findings provide evidence for a novel mechanism of gene regulation, whereby the binding of one transcription factor promotes the subsequent recruitment of another transcription factor to the same binding site. Free Full Text

Pre-activation of the genome integrity checkpoint increases DNA damage tolerance
Tsaponina O, Chabes A.
The genome integrity checkpoint monitors and reacts to defects in the genome, and it is well known that mutations in genes involved in this pathway cause sensitivity to DNA-damaging agents. Here, using budding yeast, we show that a low level of constitutive genome integrity checkpoint activation by hydroxyurea, an inhibitor of DNA replication, can increase DNA damage tolerance to multiple drugs, some of which are commonly used for cancer treatment. Modulation of the checkpoint activity may provide new strategies for utilizing this checkpoint to protect normal cells from genotoxic stress. Free Full Text

Promoter RNA Links Regulation of Inflammatory Pathways Genes
Matsui M, Chu Y, Zhang H, Gagnon KT, Shaikh S, Kuchimanchi S, Manoharan M, Corey DR, Janowski BA.
This manuscript lays out a new mechanism for using RNA to sequence-specifically recruit proteins and modulate transcription in mammalian cells. Free Full Text

Increasing The Complexity Of Chromatin: Functionally Distinct Roles For Replication-Dependent Histone H2A Isoforms In Cell Proliferation And Carcinogenesis
Singh R, Mortazavi A, Telu KH, Nagarajan P, Lucas DM, Thomas-Ahner JM, Clinton SK, Byrd JC, Freitas MA, Parthun MR.
Complexity is a critical characteristic of chromatin structure that allows for the myriad regulatory mechanisms that control cellular phenomena involving genomic DNA. The complexity of chromatin is currently thought to derive from the abundance of histone post-translational modifications and the presence of DNA replication-independent histone variants, such as histone H3.3, and histone H2AZ. Our manuscript identifies a source of complexity that has gone unexplored; replication-dependent histone isoforms. Free Full Text

Helq acts in parallel to Fancc to suppress replication-associated genome instability
Luebben SW, Kawabata T, Akre MK, Lee WL, Johnson CS, O'Sullivan MG, Shima N.
HELQ is a DNA repair enzyme that has been implicated in the Fanconi anemia (FA) pathway, a major mechanism to repair a specific type of lesion called DNA interstrand crosslinks (ICLs). Using the first reported Helq mutant mouse model, the authors show that HELQ plays only a minor role in ICL repair. Rather, HELQ was found to function in parallel to the FA core complex member FANCC for the successful completion of DNA replication and the promotion of genome integrity. This newly identified role of HELQ provides new insights into the etiology of FA, a human genetic disorder. Free Full Text

Attachment site recognition and regulation of directionality by the serine integrases
Rutherford K, Yuan P, Perry K, Sharp R, Van Duyne GD.
A family of enzymes known as the serine integrases are able to rearrange DNA molecules in the test tube, in cultured cells, and in live animals. They are particularly effective at delivering a new DNA sequence into a specific site in a chromosome, a process that has important implications for gene therapy and a variety of genetic engineering applications. This manuscript explain how serine integrases carry out these complex reactions, provides a plausible mechanism for their unidirectionality, and provide a much needed experimental framework for improving the properties and altering specificities in these systems. Free Full Text